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Crystal structure of human polynucleotide phosphorylase: insights into its domain function in RNA binding and degradation.

Lin CL, Wang YT, Yang WZ, Hsiao YY, Yuan HS - Nucleic Acids Res. (2011)

Bottom Line: The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore.Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase.Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.

ABSTRACT
Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease that degrades specific mRNA and miRNA, and imports RNA into mitochondria, and thus regulates diverse physiological processes, including cellular senescence and homeostasis. However, the RNA-processing mechanism by hPNPase, particularly how RNA is bound via its various domains, remains obscure. Here, we report the crystal structure of an S1 domain-truncated hPNPase at a resolution of 2.1 Å. The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3' tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

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Recombinant human PNPase is a trimeric phosphorylase capable of digesting RNA. (A) Domain organization of full-length (FL) and S1 domain-truncated (ΔS1) hPNPase. (B) Purity of full-length and ΔS1 hPNPase, as analyzed by 10% SDS–PAGE. (C) The gel filtration (Superdex 200) profile of full-length hPNPase shows that the enzyme was eluted as a trimeric protein. Protein markers: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa) and conalbumin (75 kDa). (D) The full-length hPNPase (0.2 µM) was incubated for 30 min at 37°C with isotope-labeled 12-mer poly(A) ssRNA. In the presence of both magnesium and phosphate ions, hPNPase cleaved RNA most efficiently.
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gkr1281-F1: Recombinant human PNPase is a trimeric phosphorylase capable of digesting RNA. (A) Domain organization of full-length (FL) and S1 domain-truncated (ΔS1) hPNPase. (B) Purity of full-length and ΔS1 hPNPase, as analyzed by 10% SDS–PAGE. (C) The gel filtration (Superdex 200) profile of full-length hPNPase shows that the enzyme was eluted as a trimeric protein. Protein markers: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa) and conalbumin (75 kDa). (D) The full-length hPNPase (0.2 µM) was incubated for 30 min at 37°C with isotope-labeled 12-mer poly(A) ssRNA. In the presence of both magnesium and phosphate ions, hPNPase cleaved RNA most efficiently.

Mentions: All PNPase from different species share similar domain structures with one α-helical, one KH, one S1 and two RNase PH domains (Figure 1A) (22). The previously reported crystal structures of two bacterial PNPases show a trimeric architecture with six RNase PH domains assembled into a hexameric ring-like conformation containing a central channel for RNA binding and degradation (23–25). The S1 and KH domains, which are presumably responsible for RNA binding, are disordered in these structures with only partially visible backbones. It was noted that the architecture of these PNPases is similar to those of exosomes which are large protein complexes participating in RNA processing and degradation in archaea and eukaryotes (26–28). The archaeal and human exosome cores contain nine proteins with six RNase PH-like proteins that form a similar hexameric ring-like structure and three S1/KH domain-containing proteins that form an S1 pore associate on the top of the ring (29–31). Current models for RNA degradation by a PNPase propose that the RNA substrates are bound by the S1 domain and/or the KH domain and are further threaded into the central channel, within which the active site is situated in the second RNase PH domain (23,26). Previous mutational studies showed that the deletion of KH and S1 domains reduces the RNA binding and cleavage activities of bacterial PNPase (24,32,33). Nevertheless, the presence of the KH and S1 domains in hPNPase seems less critical for its activity in inducing the senescence phenotype (11).Figure 1.


Crystal structure of human polynucleotide phosphorylase: insights into its domain function in RNA binding and degradation.

Lin CL, Wang YT, Yang WZ, Hsiao YY, Yuan HS - Nucleic Acids Res. (2011)

Recombinant human PNPase is a trimeric phosphorylase capable of digesting RNA. (A) Domain organization of full-length (FL) and S1 domain-truncated (ΔS1) hPNPase. (B) Purity of full-length and ΔS1 hPNPase, as analyzed by 10% SDS–PAGE. (C) The gel filtration (Superdex 200) profile of full-length hPNPase shows that the enzyme was eluted as a trimeric protein. Protein markers: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa) and conalbumin (75 kDa). (D) The full-length hPNPase (0.2 µM) was incubated for 30 min at 37°C with isotope-labeled 12-mer poly(A) ssRNA. In the presence of both magnesium and phosphate ions, hPNPase cleaved RNA most efficiently.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3351181&req=5

gkr1281-F1: Recombinant human PNPase is a trimeric phosphorylase capable of digesting RNA. (A) Domain organization of full-length (FL) and S1 domain-truncated (ΔS1) hPNPase. (B) Purity of full-length and ΔS1 hPNPase, as analyzed by 10% SDS–PAGE. (C) The gel filtration (Superdex 200) profile of full-length hPNPase shows that the enzyme was eluted as a trimeric protein. Protein markers: ferritin (440 kDa), catalase (232 kDa), aldolase (158 kDa) and conalbumin (75 kDa). (D) The full-length hPNPase (0.2 µM) was incubated for 30 min at 37°C with isotope-labeled 12-mer poly(A) ssRNA. In the presence of both magnesium and phosphate ions, hPNPase cleaved RNA most efficiently.
Mentions: All PNPase from different species share similar domain structures with one α-helical, one KH, one S1 and two RNase PH domains (Figure 1A) (22). The previously reported crystal structures of two bacterial PNPases show a trimeric architecture with six RNase PH domains assembled into a hexameric ring-like conformation containing a central channel for RNA binding and degradation (23–25). The S1 and KH domains, which are presumably responsible for RNA binding, are disordered in these structures with only partially visible backbones. It was noted that the architecture of these PNPases is similar to those of exosomes which are large protein complexes participating in RNA processing and degradation in archaea and eukaryotes (26–28). The archaeal and human exosome cores contain nine proteins with six RNase PH-like proteins that form a similar hexameric ring-like structure and three S1/KH domain-containing proteins that form an S1 pore associate on the top of the ring (29–31). Current models for RNA degradation by a PNPase propose that the RNA substrates are bound by the S1 domain and/or the KH domain and are further threaded into the central channel, within which the active site is situated in the second RNase PH domain (23,26). Previous mutational studies showed that the deletion of KH and S1 domains reduces the RNA binding and cleavage activities of bacterial PNPase (24,32,33). Nevertheless, the presence of the KH and S1 domains in hPNPase seems less critical for its activity in inducing the senescence phenotype (11).Figure 1.

Bottom Line: The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore.Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase.Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

View Article: PubMed Central - PubMed

Affiliation: Institute of Bioinformatics and Structural Biology, National Tsing Hua University, Hsinchu 30013, Taiwan, ROC.

ABSTRACT
Human polynucleotide phosphorylase (hPNPase) is a 3'-to-5' exoribonuclease that degrades specific mRNA and miRNA, and imports RNA into mitochondria, and thus regulates diverse physiological processes, including cellular senescence and homeostasis. However, the RNA-processing mechanism by hPNPase, particularly how RNA is bound via its various domains, remains obscure. Here, we report the crystal structure of an S1 domain-truncated hPNPase at a resolution of 2.1 Å. The trimeric hPNPase has a hexameric ring-like structure formed by six RNase PH domains, capped with a trimeric KH pore. Our biochemical and mutagenesis studies suggest that the S1 domain is not critical for RNA binding, and conversely, that the conserved GXXG motif in the KH domain directly participates in RNA binding in hPNPase. Our studies thus provide structural and functional insights into hPNPase, which uses a KH pore to trap a long RNA 3' tail that is further delivered into an RNase PH channel for the degradation process. Structural RNA with short 3' tails are, on the other hand, transported but not digested by hPNPase.

Show MeSH
Related in: MedlinePlus